Tumor-Derived Suppressor of Fused Mutations Reveal Hedgehog Pathway Interactions

<div><p>The Hedgehog pathway is a potent regulator of cellular growth and plays a central role in the development of many cancers including basal cell carcinoma (BCC). The majority of BCCs arise from mutations in the Patched receptor resulting in constitutive activation of the Hedgehog pathway. Secondary driver mutations promote BCC oncogenesis and occur frequently due to the high mutational burden resulting from sun exposure of the skin. Here, we uncover novel secondary mutations in Suppressor of Fused (SUFU), the major negative regulator of the Hedgehog pathway. SUFU normally binds to a Hedgehog transcriptional activator, GLI1, in order to prevent it from initiating transcription of Hedgehog target genes. We sequenced tumor-normal pairs from patients with early sporadic BCCs. This resulted in the discovery of nine mutations in SUFU, which were functionally investigated to determine whether they help drive BCC formation. Our results show that four of the SUFU mutations inappropriately activate the Hedgehog pathway, suggesting they may act as driver mutations for BCC development. Indeed, all four of the loss of function SUFU variants were found to disrupt its binding to GLI, leading to constitutive pathway activation. Our results from functional characterization of these mutations shed light on SUFU’s role in Hedgehog signaling, tumor progression, and highlight a way in which BCCs can arise.</p></div

SUFU variants in basal cell carcinoma.

<p>Whole exome sequencing with targeted re-sequencing of sporadic and Gorlin’s syndrome tumor-normal pairs revealed 9 variants, depicted in black on the SUFU gene map. Gorlin’s mutations are shown in green, while sporadic mutations are in black. Red variants are previously known SUFU mutations that disrupt SUFU-GLI1 interactions [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168031#pone.0168031.ref012" target="_blank">12</a>]. Highlighted purple regions are GLI1 binding sites. 120–125 binds the C-terminus of GLI1 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168031#pone.0168031.ref012" target="_blank">12</a>], while 388–398 binds the N-terminus of GLI1[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168031#pone.0168031.ref004" target="_blank">4</a>].</p

Select SUFU variants disrupt binding to GLI1.

<p>(A). HEK-293T cells were co-transfected with SUFU:TAP and GFP:GLI and co-immunoprecipitated to determine interaction between the two proteins. All of the truncation mutations (146X, 199X, and 464X) fully abolished any interaction to GLI1, while one point mutation (E376K) disrupted 94.9% of SUFU’s binding to GLI1, as shown by the quantification below the GLI1 blot. Asterisks indicate the correct SUFU band on the input blot. (B). Comparative growth assay in ASZ001 cells nucleofected with SUFU WT and SUFU variants. (C). E376K visualized on the known GLI-SUFU crystal structure, revealing that E376 forms a hydrogen bond to GLI1 in the pocket between SUFU’s lobes. Blue is GLI1, green is E376K, and the dashed red line is the hydrogen bond.</p

SUFU variants show differential suppression of HH signaling.

<p>(A). <i>Sufu</i>-null MEFs nucleofected with either GFP or wild type SUFU:GFP, <i>Gli1</i> mRNA is used as a readout of Hh pathway activity. (B). qRTPCR used to determine the activity of each SUFU variant, with a Western blot shown directly above to show that each protein is comparably expressed. SUFU:GFP appears as expected at 81 kDa, with truncations 146X and 199X appearing at 48 kDa and 53 kDa, respectively. Gapdh is a loading control. Asterisks denote the correct band on the GFP blot. * is P<0.005, ** is P<0.0005, *** is P<0.0001. Significance was determined by using an unpaired t test with equal SD, comparing each mutant to wild type. P values were calculated with a two-tailed comparison.</p